The invention concerns an assay for Vitamin B6, the active form of which is pyridoxal 5xe2x80x2-phosphate and to improvements in detection of H2S by using fluorescence. More specifically, the invention concerns kits and methods for determining pyridoxal phosphate concentrations in biological fluids using the apoenzyme of a homocysteinase. It also concerns improving the sensitivity of such assays and assays for homocysteine as well by measuring the fluorescence of a complex generated by the reaction of H2S with a dialkyl phenylene diamine and an oxidizing agent.
PCT publication WO 99/05311 describes high specificity homocysteine assays in biological samples using a homocysteinase enzyme with high specificity for homocysteine in comparison to cysteine sufficient for biological fluid levels. This permits the measurement of the common product, H2S, generated by these enzymes both from cysteine and homocysteine as a valid measure of homocysteine per se in physiological fluids. The H2S generated can be measured in a variety of ways as described in that application. It has now been found that the sensitivity of this assay can be improved by measuring the fluorescence of the complex formed by hydrogen sulfide with the chromogenic reagents and an N,N-dialkyl p-phenylene diamine and an oxidizing agent such as potassium ferricyanide. The resultant, 3,7-(bis dialkylamino)phenothiazine-5 chloride can be measured by absorbance or by excitation and measurement of fluorescence.
This measurement of fluorescence is also useful in the assay herein described for pyridoxal 5xe2x80x2-phosphate.
Pyridoxal 5xe2x80x2-phosphate (PLP) is the biologically active form of Vitamin B6. PLP is a cofactor for many essential enzymes involved in amino acid metabolism and fatty acid metabolism, including methioninase and homocysteinase. Additional enzymes for which PLP is the prosthetic group include glycogen phosphorylase as well as all aminotransferases. PLP is also involved in decarboxylations, deaminations, racemizations, transaminations and aldol cleavages at the xcex1-carbon atom of amino acids. These enzymes depend on PLP in order to be active, so PLP is an important metabolic factor. PLP is derived from Vitamin B6; Vitamin B6 is not synthesized by most mammals, including humans. Therefore, this vitamin is most commonly supplied in the diet. Epidemiological studies have shown that PLP deficiency is the strongest nutritional correlate to mortality from cardiovascular diseases. Deficiency of Vitamin B6 results in symptoms such as dermatitis and nervous disorders.
Given the involvement of PLP in metabolism and its deficiency in various disease states, multiple methods for the determination of Vitamin B6 levels and B6 status are known in the art.
Microbiological assays using Saccharomyces carlsbergensis (S. uvarum), Streptococcus faecium, and Lactobacillus casei have been used to measure the various forms of B6 in blood and urine. Fluorometric assays of urinary 4xe2x80x2-pyridoxic acid and blood PLP after conversion to a cyanide complex or condensation with a fluorophore, such as methyl anthranilate followed by reduction are also used. 4xe2x80x2-Pyridoxic acid can also be determined by HPLC. The PLP concentration in plasma is also measured by determining the formation of radioactively labeled tyramine from labeled tyrosine using the apoenzyme form of tyrosine decarboxylase. The reference interval is 5 to 30 ng/ml of plasma (Reynolds, R. D., Fed. Proc. (Abst. No. 2185) (1983) 42:665). This form of assay is also available commercially as an xe2x80x9cALPCOxe2x80x9d kit from American Laboratory Products Company, Ltd. (Windham, N.H. 03087).
Blood transaminases have been used as an indicator of Vitamin B6 status. The enzyme activity in serum is depressed in B6 deficiency. However, release of these enzymes reflects cell death, and breakdown in various tissues causes variability. Erythrocyte levels of aspartate and alanine aminotransferases provide a better indication of Vitamin B6 status (Briggs, M., Ed. Vitamins in Human Biology and Medicine, Boca Raton, Fla., CRC Press, Inc., 1981).
Measurement of urinary tryptophan metabolites, such as xanthurenic acid, following an oral loading (2-5 g) of L-tryptophan have also been used to indicate B6 status. Amounts of xanthurenate above the normal (25 mg/d) level indicate Vitamin B6 deficiency. A methionine loading test has also been utilized (Briggs, M., op. cit., Brown, M. L., Ed. Present Knowledge in Nutrition. 6th ed. Washington, D.C., International Life Sciences Institute-Nutrition Foundation, 1990). The ratio of cystathionine to cysteine sulfinic acid measured by amino acid analysis is elevated in a 24-h urine of B6-deficient patients after a 3-g methionine load.
Given the increasing awareness of the role of Vitamin B6 in cardiovascular disease, and the public health goal to screen patients for risk from cardiovascular disease, there is a critical need for more convenient and reliable analytical procedures that can be used to determine PLP/Vitamin B6 levels in patients. Such procedures are expected to provide considerable medical benefit both in those cases where decreased PLP/Vitamin B6 concentrations place an individual at risk for a particular disease state, and in cases where decreased PLP/vitamin B6 concentrations are a detectable byproduct of an existing disease state.
Such analytical procedures would provide great benefit by predicting a patient""s susceptibility to cardiovascular disease before onset can be detected by other procedures. In this regard, great benefit would be achieved by adapting such procedures to the widespread screening of the general population, and in particular, to patients otherwise suspected of being at risk for cardiovascular disease. It is therefore critically important that the assay be convenient to use, simple, and inexpensive. The present invention provides such methods, including diagnostic kits for use in the clinical setting.
The invention provides assays capable of detecting PLP levels in a biological sample fluids such as urine, tissue fluid, blood, whole blood, blood serum or blood plasma from a subject. The methods of the invention are thus useful to assess risk for cardiovascular disease. The invention methods also include methods of determining results which are useful not only in methods to determine PLP/B6, but also in assay methods which assess the production of H2S as a product and measure of the desired analyte using chromogenic reagents.
In one aspect, the invention is directed to a method for determining the amount of PLP in a biological sample which method comprises contacting said sample with the apoenzyme form of a PLP-requiring enzyme which can generate a product when PLP is present preferably one that is determinable by color or fluorescence. Thus, preferred apoenzymes of the invention are homocysteine/methionine alpha-gamma lyases which have been depleted of the normally associated PLP. Also preferred are such enzymes which generate hydrogen sulfide from cysteine. These enzymes generate products which are readily detectable by colorometric or fluorometric means when the enzymes are restored/activated to the holoenzyme form. The sensitivity of the assay is greatly multiplied by the function of the PLP as a co-factor; high levels of substrate can therefore be used and high levels of product generated.
In another aspect, the invention is directed to a method to improve the sensitivity of detection of H2S generated in assays, such as assays for homocysteine, which generate hydrogen sulfide, which method comprises measuring the fluorescence of the product of hydrogen sulfide with a dialkyl p-phenylene diamine and an oxidizing agent such as a ferricyanide. This improvement is especially important in assays for homocysteine or cysteine per se as the sensitivity of the assay is enhanced over one hundred fold. However, this method may also be used in assays for PLP where, because of the multiplying effect of the co-enzyme action of PLP, enhanced sensitivity is not so important.
In other aspects, the invention is directed to diagnostic kits for the methods of the invention.